	(cm)¹	Gynoecium	Stamen	Pedicel	Silique	in a pod⁴

Ws-2	34.3 ± 3.2	2.35 ± 0.02	2.51 ± 0.02	12.6 ± 1.7	13.7 ± 2.0	49.7 ± 5.1
dwf5-1	9.2 ± 1.0	1.95 ± 0.03	2.06 ± 0.02	7.9 ± 1.2	8.1 ± 0.5	46.3 ± 5.1
dwf5-2	7.6 ± 1.3	2.09 ± 0.02	1.78 ± 0.01	4.4 ± 0.7	5.1 ± 0.7	13.0 ± 3.2
dwf5-3	8.5 ± 1.8	2.02 ± 0.05	1.83 ± 0.06	7.0 ± 1.8	6.7 ± 1.6	11.3 ± 1.8
dwf5-4	11.1 ± 0.8	2.35 ± 0.02	2.06 ± 0.06	6.4 ± 0.9	8.6 ± 0.6	n.a.⁵
dwf5-5	8.3 ± 1.7	n.a.	n.a.	6.9 ± 1.1	5.8 ± 0.8	n.a.
dwf5-6	18.1 ± 0.9	n.a.	n.a.	6.3 ± 0.9	7.0 ± 0.5	n.a.
dwf4-1	4.2 ± 0.7	1.61 ± 0.04	0.99 ± 0.04	2.6 ± 0.4	3.4 ± 0.6	0.0 ± 0.0

The dwarf phenotype is typical of Arabidopsis BR dwarfs. The phenotype includes small, dark-green, round leaves, short internodes and siliques, and increased number of inflorescences. dwf5 mutants are larger than dwf4 in height with a concomitant increase in fertility. Among the dwf5 mutants, dwf5-2 and dwf5-3 are the most severe.[0188]

Key characteristics of dwf5 mutants include a short robust stature, short internodes, an increased number of inflorescences, and dark-green, round leaves similar to the phenotype previously reported for the other BR dwarfs (Azpiroz, et al., 1998; Choe, et al., 1999a, 1999b). The height of dwf5 plants was variable depending on the allele (Table 1), ranging from 22 to 53% of wild-type height at 6 weeks of age. The lengths of the petioles, pedicels, and sliliques were also reduced in dwf5 alleles.[0189]

Silique length was correlated to fertility. The longer siliques of dwf5-1 contained more seeds relative to the shorter ones of dwf5-2 and dwf5-3 (Table 1). The number of seeds in dwf5-1 siliques was the highest among the dwf mutants reported to date (Table 1; Azpiroz, et al., 1998; Choe, et al., 1999a; Choe, et al., 1999b). In addition, dwf5-1 is the only BR mutant that has been shown to possess stamens that are longer than the gynoecium similar to wild type (Table 1). In all other dwf5 alleles as well as other BR dwarf mutants, the stamens are shorter than the gynoecium.[0190]

In spite of the increased fertility in dwf5 mutants, dwf5 seeds did not develop normally. Wild-type seeds have a light-brown color and oval shape while dwf5-1 seeds have a dark-brown color and round shape. Relative to the light-brown, oval shape of the wild type, dwf5-1 seeds were darker-brown with a wrinkled morphology. The length and width of wild-type and dwf5-1 seeds is given in FIG. 1 (n=30). The average size in mm with standard deviation is shown. (Bar=1 mm).[0191]

dwf5 seeds did not germinate well compared to wild type; >90% of wild-type seeds germinated on agar-solidified Murashige and Skoog media, whereas <60% of dwf5-1 seeds germinated. This reduced germination was partially corrected by supplementing the germination media with 10[0192]⁻⁷M brassinolide. The reduced germination of dwf5 seeds may be a secondary effect of retarded growth, or possibly DWF5 is involved in seed development and/or germination.

EXAMPLE 3Biochemical Characterization of dwf5

It was previously shown that dwf7 displays reduced cell elongation, and that the short cells could be reverted to wild-type length by exogenous application of Brassinosteroids (Choe, et al., 1999b). The ability of Brassinosteroids to rescue the phenotype of dwf5 was tested. The data presented in FIG. 2 show the biochemical complementation of dwf5-1 plants with brassinolide (BL) and its biosynthetic intermediates. In the figure, the results of application of 6-deoxocathasterone (6-DeoxoCT), 22-hydroxycampesterol (22-OHCR), and brassinolide (BL) are significant increases in dwf5 pedicel length compared to a water-treated control. The length of pedicels was measured 7 days after application (n=15). Data points represent the average with one standard deviation. Further, the BR biosynthetic intermediate 6-deoxocathasterone (6-DeoxoCT), and the synthetic compound 22-hydroxycampesterol (22-OHCR) induced pedicel growth up to 80% of wild-type length.[0193]

In addition, BL application completely rescued the length of dwf5 pedicels to that of wild type, suggesting that dwf5 is deficient in endogenous brassinosteroids. Biochemical complementation by 6-deoxoCT and 22-OHCR also indicated that the defective biosynthetic step is prior to campesterol (CR). 22-OHCR has been used in feeding tests because the biosynthetic intermediates prior to campestanol (CN) were found to have undetectable activity in bioassays (Choe, et al., 1999a; Choe, et al., 1999b; Ephritikhine, et al., 1999).[0194]

Because the biochemical feeding tests suggested that dwf5 was defective in one of the steps prior to CR, the endogenous levels of CR and downstream compounds were evaluated using GC-selective ion monitoring (GC-SIM). The levels of BL and its biosynthetic intermediates were examined both in wild type and dwf5.[0195]

Scarcity of the sterols and BR biosynthetic intermediates further confirmed that the defective biosynthetic reaction in dwf5 is prior to 24-methylenecholesterol (24-MC). To pinpoint the single step disrupted in dwf5, metabolism tests were performed with[0196]¹³C-labeled mevalonic acid (MVA). FIG. 3 shows the results of biochemical analyses of the metabolism of¹³C-MVA. Important carbon atoms are numbered at the episterol structure.¹³C-labeled MVA and compactin, an endogenous MVA biosynthetic inhibitor, were fed to wild-type and dwf5 seedlings. MVA was successfully converted through the sterol biosynthetic pathway in wild type, whereas conversion is blocked at the sterol Δ⁷reduction step in dwf5. In FIG. 3, solid lines represent a single step, whereas broken lines indicate more than one step (Unit=μg/5 g fresh weight).

The basic methodologies for this experiment were described previously (Choe, et al., 1999b). As shown in FIG. 3,[0197]¹³C-MVA was converted to¹³C₅-episterol and subsequent sterols such as¹³C₅-24-MC and¹³C₅-CR in the Ws-2 wild type, but not in dwf5.¹³C₅-5-dehydroepisterol was not detected in either wild type or dwf5 possibly due to rapid conversion to the downstream compounds. Interestingly, a novel bypass pathway was created in dwf5. Unlike wild type,¹³C-MVA was converted to¹³C₅-7-dehydrocampesterol and 13C₅-7-dehydrocampestanol. Two lines of evidence, a failure to detect¹³C₅-24-MC and¹³C₅-CR in dwf, and conversion of¹³C-MVA to¹³C₅-7-dehydrocampesterol and¹³C₅-7-dehydrocampestanol possibly via^—C₅-5-dehydroepisterol, suggest that the biosynthetic steps up to 5-dehydroepisterol are active, whereas the Δ⁷reduction step is missing in the dwf5 mutants.

The results suggest that a novel pathway leading to 7-dehydrocampestanol is operating in dwf5 plants. Further details of the known components of this pathway can be found in the reference of Choe, et al., 1999b.[0198]

Experiments performed in support of the present invention indicated the following. The endogenous levels of 24-MC, CR, and CN are scarce in both weak (dwf5-1) and severe (dwf5-2) alleles. The relative ratio of 24-MC, CR, and CN to that of wild type was 2.4, 0.4, 3.7%, and 0.5, 0.2, 1% in dwf5-1 and dwf5-2, respectively. The difference in the endogenous levels of sterols in these two alleles is reflected in the degree of severity in that dwf5-2 is shorter and less fertile than dwf5-1. As a consequence of reduced sterol levels, Brassinosteroids were not detected in this analysis.[0199]

In addition to their role as precursors of Brassinosteroids, plant sterols play an important role in membranes as structural components. Membrane sterols affect the function of membrane-associated proteins by changing membrane fluidity (Bach and Benveniste, 1997). A phenotypic comparison of Arabidopsis dwarf mutants in the sterol pathway (dwf1, dwf5, and dwf7/ste1) versus those in the BR-specific pathway (det2, cpd, and dwf4) has not resulted in any obvious differences that might be attributable to a deficiency in membrane sterols. It is possible that the role of sterols e.g., sitosterol, stigmasterol, campesterol, was substituted by novel sterols, such as 7-dehydrocampesterol, or by modulating the fatty acid content in membranes. More precise analysis of dwf5 membranes e.g., ion leakage tests after heat or cold treatment, may show differences between dwf5 and wild-type membranes specifically caused by the sterol deficiency.[0200]

EXAMPLE 4Molecular Cloning and Characterization of dwf5

Experiments performed in support of the present invention (described above) indicated that the biosynthetic defect in dwf5 lies at the S7R reaction. In view of these findings, DNA sequence databases (e.g., GenBank, National Center for Biotechnology Information, Bethesda Md.; http://www.ncbi.nlm.nih.gov) were searched for candidate genes whose products were associated with the S7R reaction. An Arabidopsis S7R cDNA sequence was found in GenBank (Lecain, et al., 1996). To test if the S7R gene location corresponded to dwf5 the two markers were genetically mapped relative to each other. A cleaved amplified polymorphic sequence (CAPS) marker that was developed with the S7R gene cosegregated with dwf5, indicating linkage (0 out of 40 chromosomes).[0201]

To determine the map position of a candidate S7R gene (GenBank accession number U49398), a CAPS marker (Konieczny and Ausubel, 1993) for the locus was created and the linkage of this polymorphism to dwf5 was tested. Routine molecular techniques for DNA and RNA handling were performed according to Sambrook, et al. (1989). Some of the oligonucleotide sequences employed were as follows (sequences are given in the 5′ to 3′ direction):[0202]


DW5_FF,	GTGTGAGTAATTTAGGTCAACACAGATCA;	(SEQ ID NO:_)

DW5_LR,	GGCTCGGTCTTTTGATGATTCCAACGTT;	(SEQ ID NO:_)

DW5_2F,	TGTGGTAACCTAATAATTGACTTCTATT;	(SEQ ID NO:_)

DW5_2R,	GGAGAAGTGTAGACAGAAGGCACCCACACT;	(SEQ ID NO:_)

DW5_3F,	ATTGGAACACCATGGACATTGCACATGAC;	(SEQ ID NO:_)

DW5_4F,	AGGGTCCAATATCTCCAGCCGGAAACCGA;	(SEQ ID NO:_)

DW5_4R,	GAAAATATTTCACCCAAGTGATCATAGA;	(SEQ ID NO:_)

DW5_5F,	GGGTGCCTTCTGTCTACACTTCTCCAG; and	(SEQ ID NO:_)

DW5_5R,	AAATGACGAGCCAATCCCCA.	(SEQ ID NO:_)

In the primer designations the underlined space was used to distinguish forward or reverse primers from the gene acronym DW5, e.g., DW5[0203]_—5R. Because only a cDNA sequence was available for S7R, possible exon-intron junctions were predicted before designing primers using the RNASPL utility available at the Baylor College of Medicine (Houston, Tex.; http://www.hgsc.bcm.tmc.edu/SearchLauncher/). After sequences were determined the primers were purchased from Sigma-Genosys Biotechnologies, Inc. (The Woodlands, Tex.). Template DNA for PCR was isolated from 2 or 3 leaves (Krysan, et al., 1996). The longest PCR product spanning the whole coding region was found to be 3.5 kb in the amplification using DW5_FF and DW5_LR primers. Of the primer sets, DW5_—3F and DW5_LR yielded a 1.6 kb DNA fragment corresponding to the 3′ half of the gene. This PCR product was further analyzed to detect polymorphisms among ecotypes. One-tenth of the PCR amplification products from each ecotype was subjected to a restriction digestion with enzymes that were predicted to restrict the cDNA sequence of Columbia ecotype (GenBank Accession No. U49398). HaeIII cuts Columbia DNA once to produce 0.9 kb and 0.7 kb fragments, whereas Ws-2 and Ler DNA were unrestricted. This polymorphism was used to test for cosegregation with dwf5.

To make a mapping population, F1 seeds were obtained by crossing dwf5-1 to Columbia wild type. Fifty different dwarf plants from the F2 generation were selected for genomic DNA isolation. Individual PCR products were amplified using DW5[0204]_—3F and DW5 LR primers and the amplification products were digested with HaeIII. These restriction digestion samples were run on 1% TAE (40 mM Tris-acetate and 10 mM EDTA) agarose gel to resolve the digestion fragments and determine the restriction patterns. The mapping results indicated that the S7R gene was linked to dwf5.

DNA samples corresponding to dwf5 alleles were sequenced at the Arizona Research Laboratory (Tucson, Ariz.) using an ABI-377 automated sequencer (Applied Biosystems, Foster City, Calif.). Putative mutations were identified by comparing the sequence of mutant DNA with that of the wild-type background from which the mutant alleles were derived. The putative mutation was confirmed by repeated sequencing (>2) of independent PCR products for each strand. Sequence analysis was performed using software packages, such as Genetics Computer Group (Madison, Wis.), DNASTAR (Madison, Wis.), and utilities available at the Arabidopsis web site (http://genome-www.stanford.edu/Arabidopsis/). A summary of the genomic sequences of DWF5 and dwf5 mutants are presented in FIG. 7. FIG. 8 shows a sequence of a DWF5 cDNA and the corresponding translation product. Further, a summary of the six independent alleles of Arabidopsis dwf5 and their corresponding mutations are presented in Table 2.[0205]

TABLE 2


Six independent alleles of Arabidopsis dwf5 and their mutations.

Allele	name	Ecotype	Mutagen	Genomic DNA (4880 bp)	Amino acid (432 aa)

dwf5-1	2	Ws-2¹	T-DNA²	single bp deletion (3342³)	elongated C-terminus (+44 aa)
dwf5-2	N398⁴	En-2⁵	unknown	G2588⁵A, 3′ splice site of	stops at 298, 344, ormore
				intron
8
dwf5-3	N402⁴	En-2	unknown	C3184⁵T	Arg to stop (400)
dwf5-4	lepida	Estland	unknown	G1868⁵A	Asp to Asn (257)
dwf5-5	wm9-4	Ws-2	EMS⁶	C3211⁵T	Arg to stop (409)
dwf5-6	marial	Ws-2	EMS	G3219⁵A, 5′ splice site of	Arg to stop (411)
				intron 12

Alignments of the resulting sequences are schematically presented in FIGS. 4A and 4B. FIGS. 4A and 4B were made using the Gene Construction Kit (Textco, West Lebanon, N.H.) and box shading of the multiple sequence alignment was done using ALSCRIPT (Barton, 1993). The organization of dwf5 was determined by comparing the sequences of genomic DNA with the cDNA. FIG. 4A displays a schematic of the dwf5 gene which consists of 13 exons and 12 introns. All 12 introns were bordered by 5′-GT and AG-3′ splice site sequences. A single mutation was found in the S7R sequence from all six alleles.[0206]

The illustration in FIG. 4A shows that dwf5 contains 13 exons (filled rectangles) and 12 introns (line). The 5′(37 bp) and 3′(198 bp) untranslated regions are shown in thicker lines. The position of adenosine in the translation start codon ATG was set at[0207]position 1. The six independent mutant alleles are shown with base changes and amino acid changes in parentheses. Arrows correspond to positions of PCR primers used for RT-PCR described below.

The illustration in FIG. 4B shows a schematic view of the region from exon 9 to the 3′ UTR (based on sequencing data). The position of the stop codon is depicted with a hexagonal stop sign. The dwf5-1 mutation extended translation to add 54 aberrant amino acids and to shorten the 3′ UTR to 65 bp. Two major types of aberrant splicing patterns were identified: dwf5-2-A and dwf5-2-B chose cryptic 3′ splice sites 25 bp and 62 bp downstream of the original, respectively. These aberrant splicing patterns caused frameshifts and eventually introduced premature stop codons. In dwf5-6, the mutation was found in the 5′ splice site of[0208]intron 12. The major splicing defect in dwf5-6 was a failure to splice out the last in the mRNA, resulting in creation of a premature stop codon.

To confirm that the S7R gene encodes DWF5, the genomic DNA of Ws-2, En-2, and Estland wild types, were sequenced and compared to sequence obtained from the mutant alleles, described herein, of dwf. Genomic DNA flanking the dwf5 cDNA was isolated by thermal asymmetric interlaced PCR (Liu, et al., 1995). The following two sets of nested primers were used to amplify each of the 3′ and 5′ flanking DNAs (the oligonucleotide sequences are presented in the 5′ to 3′ orientation):[0209]


D5-3-1,	TTACTCTGATTTGCTGACAATATTCGGGTTTTG;	(SEQ ID NO:_)

D5-3-2,	GTAAAAAGGTATGGGAAATATTGGAAGCTGTAT;	(SEQ ID NO:_)

D5-3-3,	ATTGTAACGAAGTCTGTTGTTCTCATTTTCTAC;	(SEQ ID NO:_)

D5-5-1,	AGGAGCCAGAAAAGTGTGCGAGTC;	(SEQ ID NO:_)

D5-5-2,	CAGGAGAATGACGAAAGGTGGACA;	(SEQ ID NO:_) and

D5-5-3,	TGGACAGAAGGCGAGAAGCGATAA.	(SEQ ID NO:_)

Arbitrary degenerate primers, AD1, AD2, and AD3 were synthesized following the sequence described by Liu et al. (1995).[0210]

The results of these analyses indicated that the S7R gene encodes dwf5.[0211]

Based on the sequence comparisions described above (FIGS. 4A and 4B), altered splicing patterns were expected to be found in plants bearing the two alleles, dwf5-2 and dwf5-6. Evaluation of the mRNA products of these alleles was carried out by RT-PCR and subsequent sequencing of the resultant PCR products.[0212]

RT-PCR was carried out to synthesize DWF5 cDNA from the wild-type and dwf5 mutants. Total RNA was subject to DNAase I treatment (Boehringer Manheim-Roche, Indianapolis, Ind.) to remove genomic DNA. RNA was purified from the DNAase reaction using a RNEASY MINI KIT (Qiagen, Santa Clarita, Calif.). cDNA was first synthesized using reverse transcriptase (BRL, Gaithersburg, Md.) with a poly T primer called A1T17 (5′-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3′) (SEQ ID NO:______). dwf5 cDNA spanning the whole coding region was amplified using primers D5WKPN-F (5′ ATCGGTACCAAGCAGAAGAAGAAAATGGCGGAG-3′) (SEQ ID NO:______) and D5BAM-5 (5′-ATCGGATCCGCATTTTTGTTTTGGCTCGGTCTTTTGA-3′)(SEQ ID NO:______).[0213]

To amplify the possible misspliced region, A1T17-primed total cDNA was used as a template for PCR with DW5[0214]_—5F and DW5_LR primers. PCR for wild-type genomic DNA was also performed to check if there was any undesired amplification from residual genomic DNA in the RT-PCR.

One-tenth of each of the PCR amplification products was fractionated on 1.1% agarose gel, and the DNA was transferred to a membrane for DNA gel blot hybridization using dwf5 cDNA as a probe. As size controls, wild-type genomic DNA (WT-G) and cDNA (WT-C) were amplified in conjunction with RT-PCR of dwf5-2 and dwf5-6 cDNA. Two major DNA fragments in the dwf5-2 lane and one band from dwf5-6 were gel-purified using Prep-A-Gene kit (Bio-Rad, Hercules, Calif.) and sequenced to confirm the splice pattern shown in FIG. 4B. These results are further discussed in Example 6 (below).[0215]

The PCR product of the wild-type gene DWF5 was cloned into a pCR 2.1-TOPO TA cloning vector (Invitrogen, Carlsbad, Calif.), and sequenced to check for any PCR errors before subcloning into the KpnI and BamHI sites of pART7, which are designed to overexpress the cDNA under the control of the 35S promoter. pART7 is a bridge plasmid for the plant binary vector pART27 system (Gleave, 1992). This overexpression construct was designated AOD5 (Arabidopsis Overexpressor of DWF5).[0216]

EXAMPLE 5Sequence Alignment and Identification of Motifs

To examine the degree of identity between sterol reductases, as well as to validate the biological importance of dwf5 mutations in the context of the protein sequences, multiple sequence alignments were performed using the six protein sequences showing the highest similarity with dwf5 in a BLAST search (Altschul, et al., 1997). The sequences included human (Accession no. NM[0217]_—001360) and rat (AF057368) S7Rs, yeast sterol Δ¹⁴reductase (JC4057), and lamin B receptors (LBR) isolated from human (NM_—002296), rat (AB002466), and chicken (P23913). The overall amino acid identity between dwf5 and other sequences was less than 40%, with many residues being highly conserved between the sequences.

The results of the multiple sequence alignment of dwf5 with similar sequences are presented in FIG. 5. The sequence for DWF5 is shown in FIG. 8 and the sequences of the dwf5 mutants are presented in FIG. 7. GenBank accession numbers for the other sequences are as follows: NM[0218]_—001360 (S7R-human, human sterol Δ⁷reductases), AF057368 (S7R-rat), JC4057 (S14R-yeast, yeast sterol C-14 reductase), and NM_—002296 (human LBR), AB002466 (rat LBR), and P23913 (chicken LBR).

In FIG. 5, about 200 N-terminal amino acids of all LBRs and 29 C-terminal amino acids of the chicken LBR were truncated to maximize alignment. Amino acid residues conserved more than 50% among the 7 compared sequences are shown in inverse characters. Amino acid residues conserved among sterol Δ[0219]⁷reductases (S7R) are boxed. Positions for the Arabidopsis dwf5 mutations are annotated with filled triangles and described underneath. Mutations causing Smith-Lemli-Opitz syndrome (SLOS) are shown with filled circles according to Fitzky et al. (1998). “fs” refers to frameshift mutations. Introns are indicated with open circles (Arabidopsis) or open triangles (human DHCR7). Previously reported domains, EFGGxxG and LLxSGWWGxxRH, a newly identified S7R signature, and the mixed charge cluster are shaded and labeled. Dashes in protein sequences indicate gaps introduced to maximize alignment. A consensus sequence is shown in the bottom row of the alignment. Dashes in the consensus mean <50% identity among the 7 sequences compared. Capital letters stand for residues conserved among all 7 sequences, whereas lower case letters mean 50% identity. Multiple sequence alignment was performed using PILEUP in the Genetics Computer Group software (Madison, Wis.) with a gap creation penalty of 4 and a gap extension parameter of 1.

As indicated in FIG. 5, the previously reported LLxSGWWGxxRH and EFgGxxG signatures (Lecain, et al., 1996) (upper case for fully identical residues, lower case if conserved >50%, and x for variable residues) were identified at the N-terminus. In addition, it was found that the GrCLiWGrk signature is only conserved in Δ[0220]⁷reductases but missing in other reductases (FIG. 5).

Furthermore, it has been proposed that the last 40 amino acids consist of a hydrophilic soluble domain (Moebius, et al., 1998). Analysis of protein secondary sequence using the Statistical Analysis of Protein Sequence (SAPS) program (Brendel, et al., 1992) showed that this soluble domain encompasses a significant mixed charge cluster (MCC) from 399 to 426. Sixteen of the clustered 28 amino acids had either a positive or negative charge. The unusual clustering of charged amino acid residues suggests that this moiety plays an important role for proper function in this group of enzymes. In support of this, many of dwf5 mutations were directly associated with this domain by deleting part or all of it.[0221]

To summarize, the dwf5 sequence was most similar to human and rat S7R5. Yeast sterol Δ[0222]¹⁴and Δ²⁴reductases, and the C-terminal 400 amino acids of human and chicken LBR also showed significant similarity (FIG. 5). LBRs show identity with dwf5 because the 400 amino acids of the C-terminal domain reportedly possess a Δ¹⁴reductase activity (Silve, et al., 1998), whereas the remaining N-terminal domain has been proposed to be involved in nuclear assembly during the cell cycle (Gant and Wilson, 1997). The combined function of the whole protein is as yet unclear. Lecain et al. (1996) first identified two types of signature sequences in Δ⁷reductases. One group of amino acid residues, LLxSGWWGxxRH (SEQ ID NO:______) was commonly conserved in all of the sterol reductases, whereas the other group, EFGGxxG (SEQ ID NO:______), distinguished Δ⁷reductases from other sterol reductases (FIG. 5). In addition to these, our sequence alignment revealed two additional domains both located in the C-terminal half of the protein. First, a signature consisting of GrCLiWGrk (SEQ ID NO:______) was only found in S7R sequences (FIG. 5), suggesting a specific role in these enzymes. Second, an unusual cluster of charged amino acid residues was identified at the C-terminal end of all the reductase sequences. A mixed charge cluster (MCC) is highly conserved within the cut family of homeodomains proteins, suggesting an important role in this group of proteins (Brendel, et al., 1992). Similarly, it is likely that the terminal MCC domain of the sterol reductases plays a pivotal role for the proper function of the enzymes. In support of this, most of the dwf5 mutations described in the examples below were directly associated with this domain. dwf5-3 and dwf5-5 carried premature stop codons in the MCC domain, resulting in strong mutations. dwf5-4 contained a mutation changing a conserved Asp (D) to Asn (N) located at the starting region of the 6th transmembrane domain (FIG. 5; Fitzky, et al., 1998). In relation to this, many of the mutations in human SLOS patients were found in or near the putative transmembrane domains (Fitzky, et al., 1998).

Most of the sterol reductases that are involved in sterol and BR-specific biosynthesis have been isolated from Arabidopsis. These include reductases for Δ[0223]⁷(DWF5), Δ²⁴(DWF1), and Δ⁵(DET2). It has been shown that the human steroid Δ⁵reductase gene expressed using the 35S promoter in det2 could complement the mutant phenotype, suggesting that the function of Δ⁵reductases are conserved between humans and Arabidopsis (Li and Chory, 1997). However, in spite of significant sequence identity (FIG. 5), the function of the S7R was marginally conserved between Arabidopsis and humans. Ectopic overexpression of the human S7R gene in dwf5 did not induce significant complementation of the dwf5 phenotype. Furthermore, none of the intron positions were shared between the Arabidopsis and human S7R sequences (FIG. 5). Phylogenetically, in contrast to the Δ⁵reductase gene, the other plant and animal reductases appear to show independent evolution.

EXAMPLE 6Splice Site Mutations in dwf5-2 and dwf5-6

Two mutants, dwf5-2 and dwf5-6, possessed splice site mutations. dwf5-2 carried a mutation at 2588, numbered relative to the start codon (position 3258 of the sequence shown in FIG. 7), abolishing a 3′ splice site (AG-3′) by changing it to AA-3′ which resulted in aberrant splicing of[0224]intron 8. To determine the splicing patterns and their effects on translation, we performed a reverse transcriptase polymerase chain reaction (RT-PCR). Two different DNA fragments derived from aberrant splicing patterns were amplified. Comparison of the cDNA sequences from wild type and dwf5-2 revealed that two different cryptic recognition sites were selected. dwf-2-A used a cryptic site 25 bp downstream of the original one, while dwf5-2-B used one 62 bp downstream. These splicing patterns are summarized in FIG. 4B. The use of the downstream cryptic sites resulted in partial deletion of exon 9, leading to the introduction of premature stop codons. The premature stop codons created in dwf5-2 are the N-terminal-most found in the dwf5 mutants (FIG. 5), and the phenotype of dwf5-2 is the most severe among the dwf5 alleles (Table 1). In addition, lightly hybridizing bands in the dwf5-2 lane (FIG. 4B) indicated that there may be additional types of mis-splicing as minor events.

Another frame shift mutation caused by mis-splicing was found in dwf5-6. A guanosine at 3219, numbered relative to the start codon (position 3889 of the sequence shown in FIG. 7) was changed to adenosine, resulting in removal of the 5′ splice site of the last intron (5′-GT to 5′-AT). As with dwf5-2, RT-PCR analysis was employed to detect mis-spliced mRNA. The size of the transcript in dwf5-6 was larger than that of the wild-type control (WT-C lane), suggesting that the last intron was not spliced out. This was confirmed by sequencing the cDNA made from dwf5-6 transcripts. A failure to splice out the intron is predicted to create a stop codon at position 3219 (position 3889 of the sequence shown in FIG. 7), deleting half of the C-terminal mixed charge cluster (MCC; FIG. 5).[0225]

EXAMPLE 7Nonsense and Missense Mutations in dwf5-3, dwf5-4, and dwf5-5

Three mutants contained point mutations, introducing premature stop codons (dwf5-3 and dwf5-5) and a substitution of a conserved amino acid residue (dwf5-4). Position 3184, numbered relative to the start codon (position 3854 of the sequence shown in FIG. 7) in dwf5-3 was changed from cytidine to thymidine, replacing an Arg (CGA) with a premature stop codon (TGA) at amino acid residue 400 of FIG. 7. This premature stop codon resulted in deletion of most of the MCC. dwf5-4/le possessed a mutation changing guanosine at 1868, numbered relative to the start codon (position 2538 of the sequence shown in FIG. 7) to adenosine. This change resulted in the substitution of an acidic Asp (D) residue with an uncharged Asn (N) at amino acid position 257 of FIG. 7. The D was conserved in all seven peptide sequences compared (FIG. 5), suggesting a pivotal role for the D residue in this group of proteins. Another premature stop codon close to the C-terminal end, at nucleotide position 3881 of FIG. 7 (amino acid position 409) was found in dwf5-5. This mutation deleted only half of the MCC domain, but still caused a loss-of-function phenotype, suggesting that functionally important amino acid residues are present in the deleted half of the domain.[0226]

EXAMPLE 8A Mutation Affecting mRNA Stability and Further RNA Analysis

dwf5-1 contained a single base deletion (adenosine at 3343, numbered relative to the start codon; position 4012 of the sequence shown in FIG. 7) near the stop codon (FIGS. 4A and 4B). The frameshift caused by this single base deletion is predicted to result in aberrant translation of 54 amino acids before the new stop codon. See the second row of amino acids corresponding to nucleotide positions 4013-4178 of FIG. 7. Hydrophathy analysis revealed that these newly added residues created an additional transmembrane domain. 3′ rapid amplification of cDNA ends (RACE) revealed that the size of mRNA from wild type and dwf5-1 was the same. This extended translation, consequently, resulted in shortening the 3′ untranslated region from 198 bp in wild type to 65 bp in dwf5-1. As such, the dwf5-1 phenotype may possibly be due to toxicity of this novel translation product or mRNA instability caused by translation of the 3′ UTR. To further investigate this issue steady state mRNA levels were examined in mutant and wild-type strains.[0227]

To determine the steady state levels of DWF5 mRNA in different tissues, total RNA was isolated from organs of 3-week-old Ws-2 wild-type plants. Tested tissues included the shoot Apex and unopened flowers (SAF), stems, mature siliques, pedicels, rosette leaves, roots, dark-grown seedlings, and callus. Roots were taken from 10-day-old seedlings grown on vertically oriented agar plates. For dark-grown tissues, seedlings were grown in the dark for 10 days. Root-derived callus was induced and grown on callus inducing media (Feldmann, 1992).[0228]

For expression levels in wild types and mutant alleles, plants of Ws-2, En-2 wild type, dwf5-1, dwf5-2, dwf5-3, dwf5-4, dwf5-5, dwf5-6, dwf7-1, dwf4-1, bri1-5 were grown for three to four weeks. Aerial parts of the plants were harvested and frozen with liquid nitrogen. RNA extraction with the RNEASY plant kit (Qiagen, Santa Clarita, Calif.) was performed following the manufacturer's directions.[0229]³²P labeled DNA probes were made with the REDIPRIME II kit (Amersham Pharmacia, Piscataway, N.J.) using dwf5 cDNA and the tubulin α3 gene (TUA, Ludwig, et al., 1987) as templates.

FIGS. 6A and 6B present the results of RNA gel blot analysis of dwf5 transcripts. In FIG. 6A, RNA was isolated from two wild types, all of the dwf5 alleles, dwf7, dwf4, and bri1-1-5. dwf5 cDNA and an α3 tubulin gene (for loading control) were used as probes. Significantly decreased steady state mRNA levels were found in the dwf5-1 and dwf5-2 lanes.[0230]

In favor of the case for mRNA instability, the RNA gel blot analysis of dwf5 with total RNA isolated from different genotypes indicated that the steady state level of Δ[0231]⁷reductase mRNA in dwf5-1 was significantly lower than that of wild type.

To examine how DWF5 expression is spatially controlled, RNA gel blot analysis was performed with total RNA isolated from different tissues including the shoot apex and unopened flowers (SAF), stems, mature siliques, pedicels, rosette leaves, roots, dark-grown seedlings, and callus (FIG. 6B).[0232]

In FIG. 6B, total RNA was isolated from the tissues indicated, and probed with dwf5 cDNA. dwf5 was strongly detected in the SAF and roots. As a loading control, the 28S rRNA region of the ethidium bromide-stained gel is shown.[0233]

The steady state level of mRNA was highest in the SAF and roots. Stems, callus, and rosette leaves showed moderate expression of the gene. Expression in siliques, dark-grown seedlings and pedicels was comparatively low among the tissues examined.[0234]

EXAMPLE 9Molecular Complementation

Molecular complementation experiments were performed for several reasons. First, to prove unequivocally that a mutation in the S7R gene was responsible for the dwf5-1 phenotype. Second, because the human S7R gene was cloned using the Arabidopsis gene as a probe (Moebius, et al., 1998), it was of interest to test if the human gene could rescue the dwf5 phenotype. To this end, overexpression constructs were made using dwf5 and human cDNAs. In particular, the construct AOH5 includes the human cDNA sequence of GenBank Accession No. AF034544 driven by the CaMV 35S constitutive promoter. The backbone plasmid vectors for AOH5 were pART7 and pART27 (Gleave et al., 1992).[0235]

The AOH5 and AOD5 constructs were introduced into the Agrobacterium strain GV3101 by electroporation. The Agrobacterium strains harboring the constructs were introduced into dwf5-1 mutant plants by a floral dip method (Clough and Bent, 1998). Successful transformants were isolated by screening for kanamycin-resistant plants grown on agar-solidified plates containing 50 μg/ml kanamycin. Ectopic overexpression of the dwf5 cDNA (AOD5) in dwf5-1 plants completely converted the mutant to wild-type phenotype.[0236]

As compared to dwf5-1, the AOD5 plant was completely reverted to the wild-type phenotype, indicating that the single base deletion in the S7R gene is the cause of the mutant phenotype. Transgenic plants containing the human overexpression construct AOH5, while larger, were not rescued to wild type. Further, biochemical analysis of sterols in AOH5 plants showed that their levels were comparable to dwf5 controls.[0237]

Thus, novel dwf5 mutants, the DWF5 genomic sequence, as well as methods of using the same, are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.[0238]

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